Fluorescent molecules or fluorophores have many uses, including optical detection, identification, and quantification of complex biological structures such as the constituents of living cells. Fluorophores are widely used in biochemical studies and in clinical diagnoses. Many fluorophores are polyaromatic or heterocyclic hydrocarbons. Their differential partitioning among cell constituents allows one to image cellular components that are otherwise difficult to visualize.
A fluorophore will absorb a photon of energy Eex=hνex=hc/λex, where h denotes Planck's constant, c is the speed of light, and νex and λex are the frequency and wavelength of the absorbed light, respectively. A fluorophore typically stays in the excited electronic state for about 1 to 10 nanoseconds. During this time some of the absorbed energy is transferred to other molecules via collisions, and some is dissipated into the molecule's own vibrational and rotational modes. The excited molecule enters a lower electronic energy state as energy dissipates. The molecule then emits a lower-energy photon as it returns to its electronic ground state. The difference between the wavelength of absorption and emission is called the “Stokes shift” λem−λex.
The quantum-mechanical and classical processes underlying fluorescence and the Stokes shift are statistical in nature, meaning that fluorescence emission does not occur at a single wavelength, but instead occurs over a spread of wavelengths around a peak fluorescence intensity λem. Similar statistical considerations apply to absorption. Absorption occurs over a spread of wavelengths around one or more resonant wavelength peaks λres. The Stokes shift λem−λex represents the difference between the absorption resonance maximum and the fluorescence peak maximum.
It is generally preferred to have a Stokes shift large enough that overlap of the excitation and fluorescence peaks is negligible. Where this condition is satisfied, appropriately-colored optical filters, monochromators, or the like may be used to discriminate between Rayleigh-scattered incident light and fluorescence from the probe molecule. This can be an important practical experimental consideration, because illumination of the specimen can be very intense in comparison to the strength of the emitted fluorescent signal. Without the ability to sharply discriminate between excitation and emission wavelengths, scattered illumination can saturate the detector, making fluorescence measurements difficult or even impossible.
The extreme sensitivity of fluorescence techniques depends on the capacity of a fluorophore to respond to intense illumination by repeating the excitation/emission cycle very rapidly, perhaps millions of times each second. “Photobleaching” can disrupt the cycle by destroying the fluorophore, for example, when an excited fluorophore breaks apart or undergoes an irreversible chemical reaction. Another preferred property of a fluorophore is resistance to photobleaching.
Another property affecting a fluorophore's usefulness is its molecular weight. All else being equal, it is generally the case that cell membranes are less permeable to larger fluorophores. In general, lower molecular-weight fluorophores will more readily enter cellular substructures, or traverse the blood/brain barrier. On the other hand, the total number of vibrational and rotational modes available to a photoexcited dye to dissipate energy tends to decrease dramatically with decreasing molecular weight. Smaller fluorophores thus tend to have smaller Stokes shifts, which in turn can make it more difficult to resolve scattered excitation radiation from true fluorescence, especially with a low fluorescence signal. It is highly desirable to have low molecular-weight fluorophores with large Stokes shifts.
The emission and absorption spectra of some fluorophores are sensitive to their chemical environment. For instance, the presence of Ca2+ causes “fura-2” and “indo-1” dyes to fluoresce at different wavelengths, allowing them to be used for in situ intracellular Ca2+ assays. Many dyes have carboxylic acid or amine groups that undergo ionization with a pH change; these ionizations create a change in fluorescence.
Dual fluorescence is known in a number of compounds, but very few previously reported compounds have demonstrated three-color fluorescence. Multi-color fluorescence is useful in “ratiometric” techniques. The combined fluorescence intensities from the different peaks provide a measure of the total amount of fluorophore present. The intensity ratios of the peaks are an indicator of the environmental conditions to which the fluorophore is sensitive, for example pH or Ca+2 concentration. Normalization of these measurements can sometimes be helpful, as the fluorophore concentration can vary—whether randomly, or as a consequence of the process under study, or as the result of photobleaching. Monitoring fluorescence intensities and ratios at multiple wavelengths can resolve ambiguities that would exist from measurements at just a single wavelength. Ratiometric techniques have been used for purposes such as determining intracellular pH, microviscosity, flow cytometry, and confocal microscopy.
Dual fluorescence has been reported in some compounds, including 4-(N,N-dimethylamino)-benzonitrile and analogues, biaryls, benzo[c]xanthenes, 3-hydroxyflavones, hydroxy-camptothecin, 6-hydroxyquinoline-N-oxides, aromatic dicarboximides, carotenoids, and 1,3-diphenyl-1H-pyrazolo[3,4-b]-quinoline.
Another preferred property of a fluorophore is that the fluorescence should be resolvable not only from the excitation wavelength λex, but also from any fluorophores that are endogenous to the specimen.
There is also a need for new fluorophores whose fluorescence may readily be distinguished from that of existing, commercial, fluorescent probes, so that the new probe may be used concurrently with existing probes in “multiplexing” techniques, the simultaneous monitoring of different biochemical or other functions with dyes possessing different membrane permeabilities, pH sensitivities, or other sensitivities.
Dyes that are active in near infrared (NIR) wavelengths have found many uses. There is relatively little interference from endogenous absorption or fluorescence in biological samples in the near infrared. Rayleigh scattering at NIR-wavelengths is low compared to visible light scattering. NIR can penetrate tissue to a greater depth. However, there are relatively few classes of NIR dyes currently available. Those that are available include phthalocyanines, cyanines, and squaraines. There is a continuing unfilled need for novel NIR fluorescent dyes.
Some NIR dyes have been modified with various functional groups to change their properties, but adding functional groups has generally been at the expense of lower quantum yields. Modifications also generally increase the molecular weight, which can interfere with the functions of biomolecules, or with a fluorophore's ability to cross cellular or sub-cellular membranes, or with its solubility. Some modifications will cause a dye to precipitate, rendering it useless for many purposes.
Phthalocyanine and squaraine dyes in biological systems often tend to precipitate or to aggregate. Squaraines can also be chemically reactive.
Cyanine dyes possess excellent NIR properties and have high molar absorptivities, adequate fluorescence, and good photostability. However, their intrinsically small Stokes shifts can make it difficult to resolve the fluorescence emission signal of a cyanine dye from the exciting radiation, or from scattered light.
L. Lee et al., Cytometry, 1989, vol. 10, 151-164 disclosed structures for benzo[a]xanthene and benzo[b]xanthene, but did not disclose a synthesis for either, nor any use for the hypothesized compounds. This work described a synthesis for a benzoxanthene starting from 1,6-dihydroxynaphthene and other reagents. After the synthesis was concluded, NMR measurements led to the conclusion that the benzo[c]xanthene isomer was the one that had in fact been made, not the benzo[a]xanthene or the benzo[b]xanthene isomer. See FIG. 12 of the present application, in which R designates an alkyl or aryl group.
W. Fabian et al., J. Chem. Soc, Perkin Trans. 2, 1996, 5, 853-856 described the results of semi-empirical calculations on three classes of regioisomers. The authors concluded on theoretical grounds that the benzo[a]- and benzo[b]-isomers should absorb and emit at a significantly longer wavelengths than other isomeric benzo- or naphthofluoresceins The authors further suggested that these molecules might be used as intracellular pH probes. However, no source, synthetic scheme, or other method of obtaining the [a] or [b] benzoxanthene isomers molecules was described or suggested. Nor, to the present inventors' knowledge, has any other prior report described or suggested any such source, synthetic scheme, or other method for obtaining these molecules. Technically, developing a synthetic route to the [a] and [b] isomers is more challenging because the nucleophilic carbon corresponding to the path to the [c] isomer is the most electron-rich of the three potential nucleophilic carbon atom sites.
C. Murata et al., “Improvement of fluorescence characteristics of coumarins: Syntheses and fluorescence properties of 6-methoxycoumarin and benzocoumarin derivatives as novel fluorophores emitting in the longer wavelength region and their application to analytical reagents,” Chem. Pharm. Bull., vol. 53, pp. 750-758 (2005) discloses the synthesis of various 3-substituted-6-methoxycoumarin derivatives, benzocoumarin derivatives, and their fluorescence properties and Stokes shifts.
Benzo[c]xanthenes have been reported to exhibit dual ratiometric fluorescence, to have well-resolved emission bands at relatively long wavelength absorptions and emissions, and to have near-neutral pka's. They also exhibit clear isosbestic and isoemissive points. J. Whitaker et al., “Spectral and photophysical studies of benzo[c]xanthene dyes: Dual emission pH sensors,” Anal. Biochem., vol. 194, pp. 330-344 (1991) discloses a series of long-wavelength, benzo[c]xanthene dyes, their dual fluorescent emission bands, and their use in pH measurements.
C. Chang et al., “A tautomeric zinc sensor for ratiometric fluorescence imaging: Application to nitric oxide-induced release of intracellular zinc,” Proc. Natl. Acad. Sci. USA, vol. 101, pp. 1129-1134 (2004) discloses a tautomeric seminaphthofluorescein probe and its use in the intracellular, dual-emission, ratiometric, fluorescent, selective imaging of Zn2+.
Multi-color fluorescence allows one to create a range of emission colors, as perceived by the human eye. For example, equal mixing of red and green is perceived as yellow. Traditional methods for the fluorescent generation of white light generation have typically mixed different compounds emitting at three different frequencies, such as a mixture of separate red, green, and blue fluorophores.
There have been a few prior reports of single-component white-light emitters. None are closely related chemically to the novel compounds disclosed here. For example, M. Bowers et al., J. Am. Chem. Soc. 2005, 127, 15378-15379 disclose white light, broadband photoluminescence from cadmium selenide nanocrystals.
K. Hutchison et al., J. Am. Chem. Soc., 1999, 121, 5611-5612 disclose white-light electroluminescence from a fullerene adduct.
Y. Liu et al., J. Am. Chem. Soc., 2006, 128, 5592-5593 disclose white-light electroluminescence from a carbazole-substituted aromatic enyne.
W. Xie et al., J. Phys. D: Appl. Phys. 2003, 36, 1246-1248 disclose a white light-emitting device whose structure included indium tin oxide glass substrate/50 nm N,N′-bis-(1-naphthyl)-N,N′-diphenyl-1,1′-biphenyl-4,4′-diamine hole transporting layer/0.05 nm 4-(dicyano-methylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran/25 nm 4,4′-bis(2,2′diphenylvinyl)-1,1′-biphenyl/15 nm tris(8-hydroxyquinoline) aluminum electron transporting layer/0.5 nm lithium fluoride/aluminum.
W. Jia et al., Chem. Mater. 2005, 17, 164-170 disclose an electroluminescent blue emitter molecule that contains part of the N,N′-di-1-naphthyl-N,N′-diphenylbenzidine functionality, and a three-coordinate boron center. There was a substantial shift in the wavelength of maximum emission in different solvents.
P. Furuta et al., J. Am. Chem. Soc., 2004, 126, 15388-15389 disclose white-light electroluminescence from a platinum-functionalized random copolymer.
Y. Liao, J. Am. Chem. Soc., 2005, 127, 9986-9987 disclose white-light photoluminescence from microporous zinc gallophosphate.
K. Hutchinson et al., J. Am. Chem. Soc., 1999, 121, 5611-5612 disclose a white light-emitting diode fabricated by blending a Th-hexapyrrolidine C60 adduct with poly(9-vinylcarbazole) and 2,5-bis-(4-naphthyl)-1,3,4-oxadiazole.